Dying like Flies

Two papers in this issue of Cell (3xBergmann, A, Agapite, J, McCall, K, and Steller, H. Cell. 1998; 95Abstract | Full Text | Full Text PDF | PubMed | Scopus (335)See all References, 13xKaruda, P and White, K. Cell. 1998; 95See all References) indicate that a critical target of this pathway is the death-inducing protein Hid. Karuda and White used a genetic screen for mutations that ameliorate the lethality of the proapoptotic Reaper protein transgenically targeted to the Drosophila eye using the glass multimer reporter (GMR). Mutations that interfere with any component of the DER-Ras-MAPK pathway exacerbate Reaper-mediated killing whilst corresponding gain-of-function mutations suppress it. The anti-apoptotic effect of the Ras/MAPK pathway is not confined to the eye: disseminated expression of a constitutively active Ras mutant (Q13) greatly suppresses the widespread cell death that accompanies normal embryonic development in the fly. This developmental death requires genes within the 75C1-2 region of Drosophila chromosome 3 that contains the apoptotic effectors reaper, hid, and grim. Thus, the product of one or more of these genes is the initiator of cell death triggered by interfering with Ras/MAPK signaling.Subsequent analysis showed that expression of hid, but not reaper or grim, is directly modulated by Ras/MAPK signaling. Activating mutations of either Dras1 or Draf suppress hid expression, an effect mimicked by ectopic expression of Pointed P2, an Ets domain transcription factor that is functionally activated by MAPK. Conversely, both a dominant interfering mutant of the DER receptor and ectopic expression of Yan, a transcription factor that is inactivated by MAPK and which antagonizes Pointed P2, induce accumulation of hid mRNA and promote apoptosis. An important role for hid as a target for Ras/MAPK signaling is confirmed by the fact that such ectopic apoptosis is, at least partially, inhibited in hidnull mutants. hidnull mutants are also resistant to killing by ectopically expressed Reaper, which might suggest that Hid is a necessary effector of Reaper action. Curiously, however, hidnull mutants also exhibit resistance to killing by ectopically expressed hid, an observation that may indicate that Hid is lethal only above a critical threshold level, one never reached in the absence of expression of the endogenous gene.An alternative explanation might be that Hid action is also modulated by posttranscriptional mechanisms. This notion is validated by the study of Bergmann et al., who used a genetic screen designed to look directly for modifiers of eye ablation induced by GMR-directed expression of hid. Again, the initial trawl exposed a clutch of genes that implicate the DER/Ras/MAPK pathway in regulating Hid-mediated cell death: gap1, encoding a GTPase-activating protein, and sprouty and argos, which encode secreted inhibitors of DER activation. Again, loss-of-function alleles of DER, Dras1, Draf, or rolled exacerbate Hid killing, whereas the corresponding gain-of-function mutations inhibit it. In contrast to Karuda and White, Bergmann et al. find no evidence for suppression of Reaper-induced apoptosis by Ras, although both agree that Grim-induced cell death is independent of Ras signaling.In all organisms, Ras has multiple downstream effectors whose relative contribution to any Ras-dependent process may be dissected using Ras effector mutants that activate only one effector pathway at a time (Downward 1998xDownward, J. Curr. Opin. Genet. Dev. 1998; 8: 49–54Crossref | PubMed | Scopus (458)See all ReferencesDownward 1998). Bergmann et al. use effector mutants to confirm that the Raf/MAPK pathway is the major antiapoptotic pathway in the fly eye, although a weaker ancillary survival signal is mediated by the phosphatidylinositol 3-kinase (PI3-K)/Dakt pathway; the arcane Ral GDS pathway does not appear to be involved. Bergmann et al. acknowledge that the Ras/MAPK pathway affects hid expression, but concentrate on the possibility that MAPK inhibits Hid by directly phosphorylating the Hid protein. They confirm this genetically by showing that mutation of key MAPK target residues in Hid generates a protein with enhanced lethality over the wild type and that is unaffected by Ras/MAPK signaling. Thus, the DER/Ras/MAPK pathway is a critical pathway promoting cell survival that has the Hid protein as its principal target.Does this discovery have wider implications for our understanding of the regulation of apoptosis? In principle, the answer should be yes. However, there are two potential “flies in this ointment.” The first has already been alluded to—no counterpart of Hid is known in the nematode or vertebrate. There are two possibilities: either Hid is present in these organisms but has yet to reveal itself in any genetic or biochemical analysis, or Hid (and maybe Grim and Reaper too) are arthropod inventions. The remarkable overall similarities in metazoan biology makes the latter possibility unpalatable, but without any understanding of Hid function at the molecular level, it is difficult to search for analogous functions in other species. In order to place Hid, and the pathways that regulate it, into a larger context, elucidation of Hid functions is essential.The second problem lies in any interpretation of signal transduction as a linear sequence of affectors and effectors. Although initial genetic studies in the fly eye indicated a comfortingly linear Ras→Raf→MAPKK→MAPK signaling sequence, an increasing amount of data indicates that all is not so simple. Both the receptors at the apex of this pathway and Ras itself are linked to a variety of other signaling pathways, and the same may well be true of the MAPKK Dsor and the MAPK Rolled. Furthermore, flow down the Ras→Raf→MAPK pathway is subject to extensive modulation by other intersecting signaling pathways within each cell type and through control of the intensity and timing of activation (Freeman 1997xFreeman, M. Development. 1997; 124: 261–270PubMedSee all ReferencesFreeman 1997).As in the fly, the well-characterized mammalian survival signaling pathways activated by the high affinity NGF receptor TrkA and by IGF-IR also utilize Ras as a key nexus. However, although the Raf/MAPK signaling pathway suppresses apoptosis in some neuronal and myeloid cells, studies with Ras effector mutants (identical to those used in the study of Bergmann et al.) indicate that the predominant survival signal appears to route through the Ras effector PI3-K and the serine/threonine protein kinase Akt (PKB) (Downward 1998xDownward, J. Curr. Opin. Genet. Dev. 1998; 8: 49–54Crossref | PubMed | Scopus (458)See all ReferencesDownward 1998). One critical target of Akt is Bad, a BH3 protein and antagonist of Bcl2 that is inactivated by Akt phosphorylation (Franke and Cantley 1997xFranke, T.F and Cantley, L.C. Nature. 1997; 390: 116–117Crossref | PubMed | Scopus (150)See all ReferencesFranke and Cantley 1997). Although in Drosophila, Akt exerts appreciable antiapoptotic activity (Staveley et al. 1998xStaveley, B.E, Ruel, L, Jin, J, Stambolic, V, Mastronardi, F.G, Heitzler, P, Woodgett, J.R, and Manoukian, A.S. Curr. Biol. 1998; 8: 599–602Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesStaveley et al. 1998), Bergmann et al. find that it provides only a weak ancillary survival signal in the fly eye. So does this indicate a different use of pathways in fly and vertebrate? Probably not. It is clear that Ras sits at the apex of multiple effectors, each of which presides over its own suite of diverging and multifunctional signaling pathways. In such a network of pleiotropic effectors, no single cell fate is ever really controlled by any single effector (Figure 1Figure 1). Rather, the predominant outcome of Ras signaling is a result of a network of interactions between differing signaling pathways wherein some signals are reinforced while others are gated. Fate, in cells as in man, is a tangled web.Figure 1The Ras/MAP Kinase Signaling Pathway: The Legend and the RealitySee text for details.View Large Image | View Hi-Res Image | Download PowerPoint Slide